The invention relates to a cytochrome oxidase CYP1A1 specific fluorescent probe, a preparation method and applications thereof, wherein the specific probe substrate has a 4-hydroxynaphthalimide structure, and can be used for determining the CYP1A1 enzyme activity in a biological system. The CYP1A1 enzyme activity determination process comprises: selecting a 4-hydroxynaphthalimide dechloroethylation reaction as a probe reaction, and determining the activity of the CYP1A1 enzyme in various biological samples by quantitatively detecting the generation amount of the dechloroethylation metabolites per unit time. According to the present invention, the cytochrome oxidase CYP1A1 specific fluorescent probe can be used for the quantitative evaluation of the CYP1A1 enzyme activity in biological samples having different species and different individual sources, and the quantitative determination of the CYP1A1 enzyme activity in animal culture cell culture liquids and cell preparation products from different origins so as to evaluate the drug treatment capacity of the important drug metabolic enzyme CYP1A1; and the probe reaction can further be used for rapidly screening inhibitors and inducers of CYP1A1 in vitro and evaluating the inhibition ability.